Epitope analysis of cluster 1 and NK cell-related monoclonal antibodies

By flow cytometric assays, we tested the antibodies of the Second International Workshop on Small Cell Lung Cancer Antigens against 20 normal peripheral leukocytes, four small cell lung cancer (SCLC) cell lines (one classic type, and three variant type) and one gastric cancer line (KATO 3). Thirteen antibodies (Code # 4, 12, 21, 31, 34, 41, 48, 58, 60, 61, 74, 77, 82) among 98 registered antibodies showed a very similar pattern to antibody NE150, which was previously characterised as SCLC cluster 1. Since NE150 showed a positive reaction to the natural killer (NK) cell population, the serological specificity was compared with NK cell-associated antibodies, NKH1 (CD56), Leu7 (CD57) and Leu11 (CD16). Only NKH1 antibody showed a similar pattern to NE150, when tested against various target cells including SCLC lines and peripheral leukocytes, suggesting that NKH1 is a cluster 1 antibody, although it was already classified as CD56 of hematopoietic cells. By sequential immunoprecipitation, the antigen detected by NE150 antibody was depleted by preincubation with NKH1 antibody, but the reactivity of NE150 was not inhibited by NKH1 antibody, suggesting that NE150 and NKH1 detect different epitopes on the same antigen molecule. Epitope analysis was also conducted with 13 antibodies of cluster 1. Ten were found to detect the same epitope as NE150. The other three did not inhibit the binding of NE150 or NKH1, suggesting that there are at least three epitopes. Since the cluster 1 antibodies were demonstrated to detect NCAM, the present results suggest the presence of at least three epitopes on this molecule.